1. Technical Field
The subject of the present invention is a method for the immunoassay of human chromagranin A, with which it is possible in particular to assay not only chromogranin A in intact form, but also the major fragments of this chromogranin A.
Such assay may be used in particular for the diagnosis and follow-up of pathologies such as, for example, pheochromocytoma and intestinal carcinoids.
2. Prior Art
Chromogranin A (CgA) is a protein having a molecular weight of 48 kDa, a pI of 4.9, whose human form contains 439 amino acids as described by Konecki et al., 1987, in reference [1]. It belongs to the family of granins with which it shares structural and physiological similarities. CgA, like chromogranin B, shows marked inter-species preservation which presupposes a major function. CgA is largely represented in the secretagogues of endocrine and neuroendocrlne cells of which, along with the other granins, it forms one of the main components. It also acts at this level as a regulatory element of the cosecretion of other entities, such as the catecholamines in the adrenal gland.
CgA also plays an essential prohormone role via the release of active peptides, subsequent to intra-granular and extra-matricial proteolytic degradation.
In appended FIG. 1, SEQ ID NO:1 is shown of the 439 amino acids described by Konecki et al., corresponding to human chromogranin-A.
FIG. 2 shows this sequence in simplified fashion, identifying in this figure some of the peptides able to be released in man, i.e. the peptides: vasostatin/xcex2 granin, chromostatin, pancreastatin, parastatin and prochromacin. Protelysis affects the dibasic sites numbered and denoted by an asterisk in FIG. 2, which are distributed along the CgA sequence and total 10 in number in human CgA. This proteolysis has been described as a recurrent phenomenon either side of the protein ends, and it has been shown that it is specific to tissues and that it leads to substantial differences in the tissue distribution of the peptides produced. The degree of intensity and the type of proteolysis may therefore be responsible for the great variability of the fragments found in the tissues, blood circulation and urine.
Recent work by Corti et al., reference [2], has confirmed this diversity by showing that in patients suffering from pheochromocytoma there exist circulating forms of different conformation and in varying proportions from one person to another. In the same way, it has been shown, through research on the WE-14 peptide, that CgA is proteolysed in different manner in normal tissues and neoplasic tissues of the pancreatic gastro-enteral tract.
In addition to its detection in normal tissues and in corresponding neoplasias, numerous studies see a diagnostic advantage in the assay of CgA. The levels of circulating CgA are significantly high in cases of pheochromocytoma, carcinoid and endocrine pancreatic tumour. This data has been confirmed and extended to other pathologies: neuroblastoma, tumours of the gastro-intestinal tract, essential hypertension. The presence of CgA has been shown in a great number of neurodegenerative pathologies including Alzheimer""s (see reference Munoz, Lab. Invest., 1991, vol. 64, pages 826-832 [15]). Some authors have also shown that the presence of CgA in cancers of the prostate could be the sign of an unfavourable development, as in the case of kidney cancer. The assay of Cga therefore offers a major advantage.
Document U.S. Pat. No. 4,758,522 [3] describes an immunoassay of CgA in which the CgA is measured by competitive assay with radiolabelled CgA for the sites of an anti-human CgA antibody.
Syversen et al. in reference [4], have described an assay of chromogranin A with the ELISA technique using an antibody directed against a C-terminal fragment of CgA corresponding to SEQ ID NO:1 amino acids 210 to 439 and radioimmunoassay of pancreastatin.
Corti et al., in reference [2], have also described two assays of CgA using monoclonal antibodies directed against SEQ ID NO:1 amino acids 81 to 90 and SEQ ID NO:1 amino acids 68 to 70 of human CgA.
These assays therefore take into consideration some CgA fragments, but they do not permit differentiation of pathological type on the results of the assay.
Indeed, proteolysis of the molecule and the multiplicity of the circulating fragments require an assay configuration which is able to detect the majority of these entities.
The purpose of the present invention is precisely a method of CgA immunoassay which measures the levels of intact CgA and the levels of major fragments found in circulating blood.
According to the invention, the immunoassay method for human chromogranin A (CgA) present in a sample comprises the use of at least one monoclonal or polyclonal antibody which binds specifically to an epitope positioned extending from (SEQ ID NO:1) amino acids 145 to 284 from the N-terminal end of human CgA.
According to the invention, the imunoassy method for human chromograinin A (CgA) present in a sample comprises the use of at least one monoclonal or polyclonal antibody which binds specifically to an epitope positioned extending from (SEQ ID NO:1) amino acids 145 to 284 from the N-terminal end of human CgA.
To implement this assay, to the sample to be assayed is added a quantity of labelled human CgA and the antibody which binds specifically to an epitope positioned in SEQ ID NO:1 amino acids 145 to 234 of CgA, and it is then left to incubate. In this manner, competitive conditions are set up between the sample CgA and the labelled CgA for the sites of this antibody. When the assay is conducted in heterogeneous phase, using a radioelement as label, it is possible to separate the antibody-hCgA complexes formed by immunoprecipitation using an appropriate antibody. By then determining the radioactivity of the complexes formed, it is possible to determine the CgA concentration of the sample with reference to a standard curve obtained from standard samples with known CgA concentrations.
It is also possible to conduct the assay in homogeneous phase, using labelled hCgA for example and an antibody able to modify the signal emitted by the labelled hCgA.
In this assay, advantageously the antibody used is specific to an epitope positioned in SEQ ID NO:1 amino acids 219 to 234 of human CgA, or the antibody specific to an epitope positioned in SEQ ID NO:1 amino acids 145 to 197 of human CgA. These antibodies are preferably monoclonal antibodies.
To carry out this assay, the preparation of the standard samples and labelled CgA may use purified human CgA from endocrine or neuroendocrine cells of human origin, or preferably recombinant human CgA.
According to a second embodiment of the invention, the immunoassay is a sandwich-type assay using a first monoclonal or polyclonal antibody which binds specifically to a first epitope positioned in SEQ ID NO:1 amino acids 145 to 234 of human CgA, and a second monoclonal or polyclonal antibody which binds specifically to a second epitope different from the first and also positioned in SEQ ID NO:1 amino acids 145 to 234 of human CgA, one of the two antibodies being labelled.
In this second embodiment of the invention, two antibodies are used that are specific to different epitopes, positioned in SEQ ID NO:1 amino acids 145 to 234 of CgA. This assay may be conducted in heterogeneous phase by immobilizing the first antibody on a solid phase and using a second, labelled antibody.
This assay may also be conducted in homogeneous phase, using a first labelled antibody and a second antibody able to modify the signal emitted by the first labelled antibody.
By way of example, the first antibody may be specific to an epitope positioned in the sequence of SEQ ID NO:1 amino acids 145 to 197 of CgA and the second antibody may be specific to an epitope positioned in SEQ ID NO:1 amino acids 219 to 234 of CgA.
It is also possible to use in this assay a first antibody specific to an epitope positioned in SEQ ID NO:1 amino acids 219 to 234 and a second antibody specific to an epitope positioned in SEQ ID NO:1 amino acids 145 to 197 of human CgA.
According to a third embodiment of the invention, the immunoassay is a sandwich-type assay using a first monoclonal or polyclonal antibody which binds specifically to an epitope positioned in SEQ ID NO:1 amino acids 145 to 234 of human CgA and a second monoclonal or polyclonal antibody which binds specifically to a second epitope positioned in SEQ ID NO:1 amino acids 250 to 301 of human CgA, one of the two antibodies being labelled.
In this embodiment of the invention, the assay may be conducted in heterogeneous phase using a first antibody immobilised on a solid phase and a second labelled antibody, or the reverse. It is also possible to carry out the assay in homogeneous phase, using a first labelled antibody and a second antibody able to modify the signal emitted by the first labelled antibody, or the reverse.
As first antibody, advantageously an antibody specific to SEQ ID NO:1 amino acids 145 to 194 of human CgA is used, or an antibody specific to SEQ ID NO:1 amino acids 219 to 234 of human CgA.
The method of the invention is particularly advantageous for the diagnosis and follow-up of pathologies such as pheochromocytoma or carcinoid tumours, as they permit positive discrimination between the sera from patients suffering from pheochromocytoma or carcinoid tumours and normal sera.
To implement the method of the invention, conventional immunoassay methods in heterogeneous phase can be used, which permit separation of the CgA-antibody complexes from the excess labelled antibody.
It is also possible, in the method of the invention, to use homogeneous phase assay methods which do not comprise such separation.
Homogeneous phase assay methods are based on the use of an antibody-antigen pair or a pair of two antibodies which, when they are close to one another, emit a different signal from the one they emit separately.
For this purpose, it is possible to use in particular an antibody coupled to a luminescent energy-acceptor compound and an antibody coupled to a luminescent energy-donor compound.
By way of example, the luminescent energy-acceptor compound may be allophycocyanine and the luminescent energy-donor compound may be a europium cryptate such as those described in EP-A-0 321 353 [II].
One homogeneous phase assay method of particular interest is the TRACE(copyright) method described in reference [4].
In respect of heterogeneous phase assays, a solid phase is used on which the non-labelled antibody used for the assay is immobilised.
The solid phases able to be used may be of different types. For example macroscopic solid phases may be used, formed of tubes, beads or fins in polymer or other materials.
The polymers able to be used are for example polystyrene, the polyamides, polypropylene, the polyoxymethylenes and styrene copolymers.
It is also possible to use finely divided microscopic solid phases, for example powders and aggregates in polymer, protein or other materials.
Under the invention, the term xe2x80x9clabelledxe2x80x9d applied to human CgA in the first embodiment of the invention or to the different antibodies in the second embodiment, shall mean that the CgA or the antibodies have been modified by a labelling element which may for example be a radioelement, a fluorescent element, a luminescent element, an enzyme, a fluorescent chromophore, a light-absorbing chromophore, or any other ligand permitting direct or indirect quantitative measurement.
The invention also concerns monoclonal antibodies specific to an epitope positioned either in SEQ ID NO:1 amino acids 145 to 197, or in SEQ ID NO:1 amino acids 219 to 234, of human chromogranin, and immunological reagents containing one of the antibodies bound to a detectable label or fixed onto a solid phase.
These antibodies are of particular interest since they are suitable for all types of assay. They give good results not only in assays conducted with conventional labels (radioelement, etc.) but also in assays conducted with fluorescent labels of cryptate and allophycocyanine type which generally can only be used with certain antibodies.
The invention also concerns immunoassay kits for human CgA containing the above-cited monoclonal. antibodies.
It also concerns an immunoassay kit for human CgA which contains:
a first antibody, and
a second antibody, at least one of the two antibodies being an antibody specific to an epitope positioned in SEQ ID NO:1 amino acids 145 to 234 of human CgA.
According to a first embodiment of the kit, the first antibody and the second antibody are chosen from among the antibodies specific to the epitopes positioned in SEQ ID NO:1 amino acids 145 to 197 and/or SEQ ID NO:1 amino acids 219 to 234 of human CgA.
According to a second embodiment of the kit, one of the antibodies is specific to an epitope positioned in SEQ ID NO:1 amino acids 145 to 197, or in SEQ ID NO:1 amino acids 219 to 234, and the other antibody is specific to an epitope positioned in SEQ ID NO:1 amino acids 250 to 301 of human CgA.
Preferably, when the kit is intended for a heterogeneous phase assay, one of the antibodies is immobilised on a solid phase and the other antibody is coupled to a detectable label.
The detectable label is iodine 125 for example.
Preferably, when the kit is intended for homogeneous phase assay, one of the antibodies is coupled to a luminescent energy-donor compound and the other antibody is coupled to a luminescent energy-acceptor compound.
Advantageously, the luminescent energy-donor compound is a europium cryptate and the luminescent energy-acceptor compound is allophycocyanine.
It also concerns a competitive immunoassay kit containing an antibody specific epitope positioned in SEQ ID NO:1 amino acids 145 to 234.
In this kit, the antibody is preferably specific to an epitope positioned in SEQ ID NO:1 amino acids 219 to 234 or in SEQ ID NO:1 amino acids 143 to 197 of human CgA.
According to the invention, the antibodies used for the assay may be polyclonal antibodies or monoclonal antibodies.
The polyclonal antibodies may be obtained from peptide fragments of human chromogranin A obtained by proteolysis of human CgA or by peptide synthesis, by in vivo or in vitro immunisation. In this case, it is necessary to couple the peptide fragment to a vector protein such as knee ligament hemocyanin (KLH) to obtain antibodies in animal.
The monoclonal antibodies may be obtained from hybridomas using the technique described by H. Kohler and C. Milstein described in Nature, 1975, 256, pages 495-497. In this case, one or more mice immunisations are made with whole chromogranin A or with peptide fragments corresponding to the desired epitopes, associated with a carrier protein, then the spleen of the animals is taken and cell fusion is made between the splenocytes and myeloma cells. The hybridomas produced are then screened to select the clones producing the antibodies specific to the epitopes of human CgA.
Preferably, according to the invention, monoclonal antibodies are used.